Bleomycins (BLMs) are currently used under the trade name Blenoxane(R), in the treatment of squamous cell carcinomas, Hodgkin's lymphomas, testicular carcinoma and in other combination chemotherapy. This application proposes to investigate the genetic and biochemical basis of the production of BLM in Streptomyces verticillus ATCC 15003 as the first step of attempting to synthesize a variety of complex anticancer drugs by a novel, economical biological system. This proposal is based on the hypothesis that BLM is a natural hybrid metabolite of peptide and polyketide biosynthesis and the BLM peptide/polyketide backbone is assembled by the BLM synthetase that should bear the characteristics of both peptide synthetase (PTS) and polyketide synthase PKS). Two corollaries of this hypothesis are that the technology developed for genetic engineering of PKS and PTS could be applied directly to manipulate the blm synthetase genes for BLM analog synthesis and that understanding the biosynthesis of BLM should shed light on the mechanism of how PTS and PKS could be integrated into a biosynthetic machinery to make complex natural products. The ultimate goal of the proposed research is to construct a hybrid PKS/PTS enzyme system for the production of hybrid polyketide/peptide metabolites. The specific goals for the five year grant period are as follows. (1) Isolate biosynthetic intermediates of BLM from S. verticillus and establish high performance liquid chromatography conditions for their analysis. (2) Confirm the already cloned DNA representing the blm biosynthesis genes by either gene disruption or replacement experiment or demonstration of BlmORF1 as the P-6m hydroxylase. (3) Identify relevant genes for BLM biosynthesis by extending sequence analysis into the adjacent regions of the sequenced 7.2 kilobase DNA fragment and map the entire blm cluster by hybridization of a genomic library of S. verticillus with the cosmid clone containing the above DNA. (4) Establish the functions of cloned genes by expressing them individually followed by biochemical and enzymological characterizations or by expressing them collectively followed by screening for BLM biosynthetic intermediate. The outcome of these studies could potentially lead to the discovery of new anticancer drugs or impact directly on the production of BLM at lower cost through modifications of the BLM biosynthetic genes, and will lay the foundation for rational designing of hybrid PKS/PTS enzymes from other peptide and polyketide pathways for the biosynthesis of novel classes of natural products from amino acids and short fatty acids.